Evaluation of healing progression at surgical incision sites and the use of antiseptics for enhancing post-operative survival in subyearling Chinook salmon (Oncorhynchus tshawytscha)

In an attempt to develop more effective surgical implantation methods for fish, surgical incisions typical of those made for implanting micro-acoustic transmitters into the peritoneal cavity were evaluated on a weekly basis for healing progression using a suite of metrics. Additionally, four chemicals were evaluated at concentrations commonly used in aquaculture for their ability to prevent surgical site infection and thus to promote incision healing and survival. Chemical treatments included hydrogen peroxide (25, 50, and 100 mg l-1), salt (10 and 30 ppt), Argentyne (1:1, Argentyne:water), and PolyAqua (1/2 tsp 36 l-1). For all study fish, the presence of two intact sutures seven days post-surgery (generally associated with good suturing technique) was negatively correlated with survival. A generalized linear mixed effects model indicated that suture presence, increasing tagging temperature, and the presence of foreign material on sutures decreased survival by 0.56, 0.72 and 0.60 respectively. Conversely, evidence of suture tearing and increasing fork length at tagging increased survival by 0.24 and 0.17. The antiseptic treatments tested promoted neither faster healing of surgical incisions nor higher survival for fish held for 28 days compared to a reference group and two of the chemicals may be contraindicated for prophylactic use at published doses. These findings underscore the need for researchers to adopt a decidedly cautious approach to planning and interpretation of study outcomes that rely on telemetry tagging, carefully considering the study subjects, potential effects of the techniques used, and implications of the environmental conditions experienced.

Introduction Aseptic techniques are practiced in human and veterinary medicine as a routine component of any surgical procedure to prevent contamination and subsequent infection [1]. More recently, such techniques have been applied in fishery research for surgical implantation of transmitters and data loggers [2][3][4][5][6][7][8][9][10][11][12]. However, given the difficulty of maintaining asepsis in aquatic surgical and post-operative environments, some researchers have expressed doubt that any benefit is obtained through their use [13].
In 2007 and 2008, a large tag-comparison study to evaluate the effects of surgically implanted acoustic transmitters on yearling and subyearling Chinook salmon (Oncorhynchus tshawytscha) was conducted within the Columbia River (CR), USA [14]. During said study, survival and behavior of fish implanted surgically with acoustic transmitters was compared to that of those implanted with PIT-tags using an injection method. For the field component of the study, fish were released back into the CR to resume their migration after recovering from anesthetic. For surgical implantation of acoustic transmitters, aseptic technique was practiced to the extent practically possible. For example, all surgical tools were sterilized in a steam autoclave prior to the start of each tagging day. Tools were rotated and reused between surgeries within the course of a day only after they were disinfected in 70% ethyl alcohol for a minimum of 10 min and then rinsed in distilled water. Surgical needles and sutures were similarly disinfected between surgeries. Despite these practices, during 2007, tagged subyearling Chinook salmon that had been intercepted downstream after release were observed to have considerable inflammation at incision sites and some exhibited gaping wounds in the vicinity of their incision.
Based on these observations, the field study was postponed, and additional laboratory research (described here) was conducted to determine the cause(s) of these observations and to explore more effective implantation methods. Towards this end, surgical implantation surgeries were performed on run of the river subyearling Chinook salmon captured at Lower Granite Dam during their outmigration from June 3 through July 16,2008. These fish were then transported to the Juvenile Fish Facility at Bonneville Dam where their surgical incisions were evaluated on a weekly basis for progression of healing using a suite of metrics (Table 1). Additionally, the efficacy of several chemicals (applied topically) was tested for preventing infection and/or promoting healing of the post-operative incision sites [15][16][17]. The underlying hypothesis was that faster and cleaner incision healing (i.e., better tissue apposition, less inflammation, and no infection) would be correlated with higher survival.
Chemical treatments tested included hydrogen peroxide (PeroxAid; Eka Chemicals, Marietta, GA), salt (Instant Ocean; Spectrum Brands, Madison, WI), Argentyne (Argent Aquaculture, Redmond, WA), and PolyAqua (Kordon, Hayward, CA) (Table 2A and 2B). These chemicals were chosen based on their current use by the aquaculture industry as either antimicrobials/parasiticides/fungicides (hydrogen peroxide and Argentyne), an osmoregulatory enhancer (salt) or a water conditioner (PolyAqua). Apart from Argentyne, the chemicals tested have not been routinely used as antiseptics during fish implantation procedures and to the authors' knowledge, a rigorous comparison of this nature has not been conducted.

Ethics statement
This research was conducted following the U.S. government principles for utilization and care of vertebrate animals used in testing, research, and training as described by the National Institutes of Health, Office of Laboratory Animal Welfare [66]. The care and use of experimental fish complied with the animal welfare laws, guidelines and policies of the United States and states of Oregon and Washington, including the Endangered Species Act of 1973, as amended, and lawful regulations issued thereunder which relate to threatened or endangered plant or animal species. The determination of take and handling for these research purposes was federally approved by National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Authorization and Permits for Protected Species, under the Federal Columbia River Power System's 2008 Biological Opinion. Similarly, the scientific collection permit for fish and marine and freshwater invertebrates was approved by Oregon Department of Fish and Wildlife, as was the scientific collection permit for state and/or federal threatened or endangered species of marine and freshwater fish, by Washington Department of Fish and Wildlife.
Useful information was sought after in terms of an antiseptic or chemical that might help surgical incision site healing and promote species conservation. No adverse effects were anticipated from applying the treatments tested to the incisions of study fish as all chemicals tested were chosen based on their use as therapeutics or water conditioners in the aquaculture Table 1. List of metrics used to evaluate incision site healing for dip study fish that survived the 24-h post-tagging recovery period and transport to the Bonneville fish facility.

Suture tearing
Evidence of tissue torn by suture 0 = absent, 1 = present

Inflammation
Presence of inflammation at suture entry or exit sites or between entry and exit sites 0 = none, 1 = present at either the suture entry or exit site or in between, 2 = present at two or more suture sites including the suture entry and exit sites and/or in between Ulceration a Presence of ulceration at suture entry or exit sites or between entry and exit sites 0 = none, 1 = present at either the suture entry or exit site, 2 = present at both entry and exit site, 3 = present between entry and exit, 4 = present at entry/exit sites and in between

Incision healing
Degree to which incision is healed 0 = completely healed, no scar visible, 1 = healed but scar still visible, 2 = incision not healed

Incision apposition
How well the two parallel sides of the incision are approximated 0 = perfect apposition on 100% of incision length 1 = >50% of incision length perfectly apposed 2 = <50% of incision length perfectly apposed

Incision inflammation
Extent of inflammation along length of the incision by percentage 0 = none, 1 = inflammation on �25% of incision length 2 = inflammation on 26-49% of incision length 3 = inflammation on >50% of incision length Remaining number of "throws" is not reflected here. a Does not describe degree of ulceration, but only whether underlying dermis is exposed and red. https://doi.org/10.1371/journal.pone.0288056.t001

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Evaluation of surgical incision healing and antiseptic use in Chinook salmon (Oncorhynchus tshawytscha)  [14,45] for a map of the study area, a detailed description of river conditions during fish collection, and for details regarding fish transport.

Fish collection and tagging
Migrating hatchery and wild subyearling Chinook salmon were diverted from the smolt collection facility at Lower Granite Dam (Rkm 173 on the Snake River in Washington State) over 10 dates between 2 June and 15 July, 2008 for this study. These fish were routed to a concrete raceway for holding and sorted within 12-18 h of collection. Sorting was conducted under light anesthesia with clove oil first, followed by tricaine methanesulfonate (MS-222; [67,68]). Approximately 400 fish per day (3200 total) were selected for tagging. These fish measured at least 95 mm in fork length, had no visual sign of disease or injury, and had not been previously implanted with PIT-tags. After sorting, study fish were transferred to a 975-L tank with flow-through river water, where they were held overnight to recover from anesthesia.
On the day after collection, study fish were anesthetized in a bath containing MS-222 in concentrations ranging from 50 to 80 mg l -1 [14,45]. After reaching stage-IV anesthesia (loss of equilibrium; [69]), fish were removed from the anesthetic bath and transferred in water to a data station where they were weighed, measured, photographed, and assigned to chemical treatment groups and surgeons. Assignments were made following a predetermined rotation. This ensured that on a given day, there was equal contribution among the surgeons present to each treatment group. The goal was to tag 40 fish for each treatment group every day for ten replicate days, totaling 400 fish per treatment for the study overall (Table 3). Sample sizes were determined based on availability of fish from the parent study [14] and the number of fish four surgeons could perform surgery on within a day. Study fish were equally balanced between the replicate days and between treatments. These sample sizes granted approximately 80% power to detect a 10% difference between the treatments. The treatment groups tested are shown in Table 2A, and included hydrogen peroxide (25,50, and 100 mg l -1 ) and salt (10 and 30 ppt), both following recommended concentrations [35,70]; Argentyne (1:1, Argentyne:water), following the recommended concentration [71]; and PolyAqua (1 tsp 37.8 l -1 ), following the manufacturer's instructions for use [59]. A reference group subjected only to a river water bath after tagging was also included. The reference group was considered to be another treatment group-i.e., not a "control" group-and analyses herein are designed to compare eight treatments.
From data stations, treatment fish were transported to the assigned surgeon, transferred from their container to a surgical platform made of closed cell neoprene and maintained on an anesthetic drip over their gills of MS-222 (50 mg l -1 ) for the duration of the procedure. A 2-3 mm skin incision was made on the ventral midline of each fish just anterior to the pelvic girdle with either a 3.0-mm Micro-Unitome blade (BD Medical Supplies: Franklin Lakes, NJ), a number 10 scalpel blade, or a combination of both. The choice of blade used or combination thereof was based on surgeon preference. This incision procedure and location was typical of the procedure used to surgically implant micro-acoustic tags in juvenile salmon. However, instead of an acoustic transmitter, a PIT tag (Biomark; Boise, ID, Tx-1411SST) was inserted into the peritoneal cavity to monitor incision healing of individuals throughout the study. On average, PIT tags measured 12.48 mm in length and 2.07 mm in diameter and weighed 0.1 g in air. Each PIT tag is characterized by a unique hexadecimal code that can be scanned and read through the body wall of a tagged fish.
Incisions were closed with 2, 5-0 absorbable monofilament sutures in a simple interrupted pattern. Surgical tagging occurred simultaneously at four stations, with approximately 75-100 PIT tags implanted per hour for a total of 320 fish tagged daily. All surgical tools were sterilized in a steam autoclave prior to the start of each tagging day. All PIT tags were disinfected in 70% ethyl alcohol for a minimum of 10 min and then rinsed in distilled water prior to use. Suture material and surgical tools were disinfected and rinsed in the same manner between consecutive surgeries.
In total, 3198 subyearling Chinook salmon were tagged for this this study and subjected to one of eight treatments (including a reference group; Table 3). For the first eight replicates evaluated, eight primary surgeons implanted PIT tags in a total of 2,297 bath treatment and reference fish (range 127-451 each). These eight primary surgeons attended from two to seven replicate days, when five of the eight generally tagged~64 fish each. Four other surgeons tagged a total of 262 fish (range 34-99 each) over the 8 replicates. Each surgeon attended 1-2 replicate tagging sessions and marked 4-66 fish.

Treatments
Immediately after surgical tagging, fish were transferred in water to a second data station, and their incision was photographed. At this stage, all study fish except the Argentyne treatment

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Evaluation of surgical incision healing and antiseptic use in Chinook salmon (Oncorhynchus tshawytscha) group were placed by treatment cohort (N = 40 fish) into 5-gal buckets supplied with flowthrough river water to recover from the anesthetic. For the Argentyne treatments, a disinfectant solution of 1:1, Argentyne:water, was applied to incisions prior to placement of fish in recovery buckets. Argentyne is composed of polyvinylpyrrolidone iodine, and was applied topically, as opposed to the other treatments, which were administered via immersion bath. For bath treatments, the last fish implanted with a PIT tag was allowed to recover from anesthesia for a minimum of 10 min before netting them from recovery buckets with soft fine mesh dip nets and distributing them to their respective treatment bath. Chemical treatments were prepared by adding premeasured amounts of each chemical and 36 L of fresh river water to pans of dark blue, nonporous polyethylene measuring 66.0 × 45.7 × 25.4 cm (Polylewton; U.S. Plastic, Lima, OH). Reference and Argentyne treatment groups were subjected to baths filled with fresh river water only. There was one bath prepared for each treatment and all were aerated throughout the treatment.
Salt baths were made using Instant Ocean to prepare solutions of 360 g and 1,080 g salt 10 l -1 river water for the respective 10 and 30 ppt treatments. Salt slurries were mixed at least 4 hours prior to use to allow for maximum dissolution. Just prior to treatment, slurries were transferred to treatment pans, and fresh river water was added to bring the entire volume up to 36 L. Salinities were then measured using a handheld refractometer, and additional solute was added in 100-g increments as needed to bring them up to 10 and 30 ppt. Salinity levels were also verified using a multi-parameter water quality meter (h19828; Hanna Instruments, Woonsocket, RI).
Water conditioner treatment baths were prepared by adding 1/2 tsp of PolyAqua to 36 L of river water. This was lower than the recommended maximum label dose of 1 tsp PolyAqua 37.8 l -1 water for ease and consistency of measurement. Reference fish were exposed only to a river water bath.
For each group, treatment time started once the last fish of the group had been transferred to the bath. Treatment duration for all but the 30 salt ppt group was 30 min. The duration of the 30 salt ppt treatment was 10 min [35]. The time of entry between the first and last fish transferred for each group was less than one minute.
Temperature, pH, and dissolved oxygen (DO) were measured in each treatment bath at 0, 15, and 30 min (30 ppt salt bath, 0 and 10 min) using the Hanna meter. Total dissolved solids were also measured in the reference bath at time zero using the Hanna meter, as was total water hardness, which was measured using water quality test strips (SofChek; Hach Company, Loveland, CO). The Hanna instrument was recalibrated weekly for conductivity (80,000 μS cm -1 ), pH (3 point; 4.01, 7.01, and 10.1), and DO (% saturation), according to manufacturer's recommendations.
Fish behavior was monitored every 5 min during treatment, with observers noting any deviations from normal swimming behavior and spatial distribution. Normal swimming behavior was indicated by calm but continuous swimming, as opposed to resting on the bottom, jumping, or gulping air at the surface. Normal spatial distribution was indicated by even distribution throughout the water column as opposed to aggregation at or near the bottom or at the surface.
Upon removal from treatment baths, fish were divided equally between one of two 75-L (19.8 gal) stainless steel holding tanks supplied with flow-through river water. Fish remained in holding tanks before being transported by truck 12-24 h later, depending on when the truck was loaded, to the Bonneville Dam juvenile fish monitoring facility in North Bonneville, WA (Rkm 235 on the Columbia River). Mean transport time was 6 h 14 min. Jugs containing frozen river water were added to the transport tank during transports to keep water temperatures within 1˚C of the temperature at departure. This method proved successful for the first four tagging dates as well as for the sixth, ninth, and tenth date. However, for the fifth, seventh, and eighth dates, temperature during transport varied from 3-5˚C above that at departure from Lower Granite Dam [14]. Fish that died within 24 h of tagging as well as those that did not survive transport, were excluded from the study.

Laboratory holding, observation, and incision evaluation
Upon arrival at the monitoring facility, fish were water-to-water transferred to 1,893-L (500-gal) circular tanks and held by date tagged (or replicate group) for the remainder of the study. Initial stocking density was less than 2 kg m -3 and remained low throughout the 28-d holding period. Study tanks were supplied with flow-through river water from the CR, and fish were fed ad libitum, a diet consisting of a mixture of appropriately sized semi-moist pellets (BioDiet Grower; BioOregon, Hammond, OR). Holding tanks were checked daily for mortalities which were removed and recorded. All surviving fish were anesthetized, examined, and evaluated for progression of incision healing on days 7, 14, 21, and 28 post-tagging based on a suite of metrics (Table 1). During this process, right-and left-lateral, full-body photographs were taken of each fish, as well as a close-up photograph of each incision (S1 Fig shows examples of metrics used to evaluate healing progress). After being held for 28 d, survivors were released to the river to resume migration.

Statistical evaluations
Sample size and means for mortality by treatment at 28 d were compared among surgeons, as well as among replicates. Mortality rates were not compared to draw definitive conclusions about individual surgeons because they were not consistently present throughout the study period. Rather, attempts were made to verify that treatment comparisons were not biased by differences in survival among surgeons.
The relative effects of bath treatments were evaluated four ways. First, to assess overall survival effects, cumulative survival was compared by treatment group at day 7, 14, 21, and 28. This comparison used two-factor analysis of variance (ANOVA), where a replicate "time block" was considered as a random factor and bath treatment as a fixed factor. Although the error structure of survival data was presumed to be binomial, it was also presumed that with the samples' sizes (n > 30 for each treatment/replicate and 10 replicates) and the bulk of the values in the range > 0.40, that the data was approximately normally-distributed. Following a significant ANOVA test, Tukey's Honestly Significant Difference (HSD) comparison test was used, which maintained the desired significance level as there were eight treatment means [72]. For this, and subsequent analyses, a significance level of α = 0.05 was established.
Second, nonparametric Kaplan-Meier "time-to-event" curves were plotted to informally examine survival patterns over time [73,74]. Because fish were released after 28 d, data from greater than 28 d were right-censored. Kaplan-Meier curves were plotted for replicates pooled across treatments to assess seasonal survival patterns, and for treatments pooled across replicates to assess within-study 28-d survival patterns.
Third, survival to 28 days was compared between incision metric scores measured at day 7 in a three-factor ANOVA (see first analysis above). Once again replicate "time block" was considered as a random factor, and bath treatments (eight) and incision metric score (2-3 levels) as fixed factors. As above, although the error structure of survival data was presumed to be binomial, it was also presumed that there was sufficient replication to treat the data as approximately normally-distributed. Some incision metric scores were combined to avoid small sample problems when relatively few fish were scored at those levels. Following ANOVAs indicating significant differences for fixed factors, multiple comparisons using Tukey's HSD were conducted.
Finally, for fish that were alive at 7 d, a logistic regression mixed-effects model (GLMM) using a logit link function for analysis of survival to 28 d was run [75]. For this model, both the tagging replicate and the surgeon who conducted the PIT tag implantation surgery were included as random effects (i.e., the model intercept was different for each replicate and surgeon), and the fixed effects included treatment, incision metrics measured at 7 d, tagging temperature, fork length, and weight.
The best model was determined using Akaike's Information Criterion (AIC) rankings [76] in three stages. First, the best model using the "measured-at-tagging" variables (see above) was identified. Next, the 7-d incision metrics (i.e., fungus present, total sutures present, sutures tearing, suture site inflammation, suture site ulceration, and incision healing) were modeled to see if the initial best model could be refined further. For this stage, possible variables from the nine shown in Table 1 were reduced to six. Total knots present was removed owing to its high correlation with total sutures present, and incision apposition and incision inflammation were removed because each had large numbers of missing values. Finally, interactions between included variables were modeled to check for further improvement. The R program was used for all analyses [77], and specifically the package MuMIn, R package version 1.43.15 for mixed-effects modeling.

Surgeon effect and environmental conditions
The number of fish tagged by each surgeon in each replicate was spread equally across treatments, so differences among surgeons did not bias comparisons of mean survival (S1 Table). However, examination of the data revealed trends in survival related to tagging personnel among both primary surgeons and those who tagged periodically throughout the study period). Marginal means for mortality ranged 28.2-54.8% among surgeons and 21.0-66.8% among replicates. Individual surgeons were not compared definitively, as they were not present consistently throughout the study. However, survival to 28 d ranked consistently among surgeons within individual replicates, thus revealing that some surgeons had higher survival than others (S2 Table).
The mean number of sutures remaining at 7 d by surgeon was also examined. This examination showed that surgeons with high suture retention (1.9-2.0 sutures), associated with more concise, secure knots/ligatures, also had lower survival at 28 d than their cohorts with lower mean suture retention (1.1-1.3 sutures; S2A and S2B Fig).
With respect to environmental conditions experienced by study fish during bath treatments, for all replicates and treatments, the DO generally increased from the beginning (mean = 9.8 mg dl -1 ; range = 7 to 19 mg dl -1 ) to the end (mean = 12.8 mg dl -1 ; range = 7.44-23.0 mg dl -1 ). For all but a few replicates and treatments, the pH dropped only slightly from the beginning (mean 7.9) to the end of treatment (mean 7.3). The pH dropped below 7.0 during replicates 6 (peroxide, salt at 10, and PolyAqua), 7 (reference, peroxide, Argentyne, and PolyAqua) and 9 (reference, Argentyne, and PolyAqua). Tagging temperature did not vary by more than 0.5˚C throughout a given day. However, temperature increased from 11 to 18˚C, from early to late replicates. Isolated jumping, and some flashing, nosing, and gulping were observed in all treatment groups at some point during the study. However, there was no evidence of group behavior having been influenced by a specific treatment or environmental parameter such as acidic pH levels.

Fish removed from the survival analyses
As previously mentioned, fish that died prior to ponding at Bonneville Dam (i.e., within the first 24 h post-tagging) were excluded from survival comparisons. This early mortality was low overall and was not equal among treatment groups (S3 Table).
Twenty-four-hour post-treatment mortality was highest for the three peroxide treatment groups, ranging 2.8-8.2% over all replicates combined, and was less than or equal to 1.5% for all other treatments. Overall, mortality prior to ponding was highest for replicates 7 (10%) and 10 (6.6%) tagged on 27 June and 16 July, respectively, and was below 3% for all other replicates (Table 4). Notably, more than 80% of all treatment fish from replicates 9 and 10 died very soon after transfer to the monitoring facility. Therefore, fish tagged on these dates were also omitted from the survival comparisons by treatment. For the modeling analyses, only 24-h mortalities and fish tagged for replicate 10 were omitted.

Survival
Percent survival by treatment and replicate at 7, 14, 21, and 28 d is detailed in Table 5. Overall, there were significant differences in survival identified at 7 d (F = 2.61, P = 0.023). However, the Tukey comparison tests showed that there were differences between replicate days but no significant differences between the bath treatments. By 14, 21, and 28 d there were no significant differences in survival identified between replicate days or between treatments (F = 1.74, 1.81, and 1.36; P = 0.122, 0.107, and 0.244). Diagnostics showed a good model fit for ANOVA, with high R 2 values (range .83-.92 for the four comparisons).
For individual days-replicates, non-parametric Kaplan-Meier curves of mortality showed increasing patterns through time (Fig 1). This pattern was also like that observed in a longterm holding study (that was run in tandem) wherein mortality was lowest in the first two replicates and highest in the last two [14,45]. Kaplan-Meier curves describing mortality by treatment showed similar temporal patterns of somewhat constant mortality (Fig 2). Table also shows the number of fish that survived transport to the Bonneville Juvenile Fish Facility (Ponded at Bonneville) and the temperature on holding day 28 corresponding to each replicate group.

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Evaluation of surgical incision healing and antiseptic use in Chinook salmon (Oncorhynchus tshawytscha)

Incision evaluations
Analysis of Variance testing indicated that there were no significant differences in survival to Day 28 among bath treatments for fish alive and scored for incision metrics on Day 7 ( Table 5). As such, all results and comparisons below between levels of incision metric are averaged across treatment groups, effectively increasing replication to 8 replicates x 8 treatments (i.e., n = 64). For fish observed at 7 d with sutures intact, survival was significantly lower at 28 d (by 0.12) compared to those with only one or zero sutures intact (Tukey HSD adjusted P = 0.002 or P = 0.004, respectively). Survival for fish with one or zero sutures intact was not different (Tukey HSD adjusted P = 0.998). Evidence of suture tearing at 7 d was positively correlated with 28 d survival (P = 0.002). Fish with torn sutures had 0.08 higher survival than those without evidence of tearing (Table 5). There was no significant relationship between 28 d survival and the presence of fungus on sutures, inflammation, ulceration, or incision healing metrics as measured at 7 d (Table 6).

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Evaluation of surgical incision healing and antiseptic use in Chinook salmon (Oncorhynchus tshawytscha)

Survival modeling
For fish alive at 7 d, the first stage in modeling 28-d survival showed that the best model included only tagging temperature and fork length at tagging (Table 7A and 7B). The next three best models were between 1 and 2 AIC units larger, and all included fork length. One was only fork length and the other two included weight, which was quite correlated with fork length (r = 0.76) and thus adding minimal information. Therefore, the model that included only temperature and fork length was used in further modeling. The second stage of modeling showed the best model included tagging temperature and fork length as well as total sutures present, foreign material present, and suture tearing (Table 7A and 7B). Although a couple of models that included ulceration or inflammation had delta AIC values below 2, they were expanded versions of the best model and did not add significant information.
From the third stage evaluation of interactions between the variables included in the stage two best model, seven combinations of interaction terms, including none, were plausible, with ΔAIC values between 1 and 2. However, none of the interaction models substantially improved the stage two best model, so following the principle of parsimony [76], the no-interaction model was chosen as the best model (Table 7A and 7B). All of the models accounted for random effects of replicate tagging days and surgeon. It is important to note that none of the best models included bath treatment. This is because it was not identified as a significant factor when tested with fork length and water temperature at tagging. As such, similar to ANOVA testing, modeling did not identify significant differences in survival related to bath treatment.
Overall estimated 28 d survival for fish that were alive and observed at 7 d increased from 0.60 to 0.75 as fork length increased from 100 to 140 mm (Fig 3). Conversely, estimated survival decreased from about 0.77 to 0.60 as water temperature at tagging increased from 11 to 16 C. For fish with one or zero sutures remaining at 7 d post-tagging, estimated 28 d survival for fish alive and observed at 7 d averaged around 0.75, but this estimate decreased to 0.60 for fish with two sutures present. When foreign material was not present at the incision site at 7 d, estimated 28 d survival averaged 0.75. In comparison, estimated average survival was only 0.65 when foreign material was present. Finally, when there was no tearing observed at the suture site on day 7, survival averaged 0.63. However, when one or two sutures were observed to be tearing through the dermis, estimated survival was higher at 0.67.

Discussion
This study set out to evaluate the healing progression of surgical incisions in subyearling Chinook salmon. Motivated by observing incision dehiscence in migrating fish surgically tagged the previous year, the main goal was to gain insight into the etiology behind these incision failures. An additional goal was to identify a treatment that might promote healing.

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This study found that mortality in some study fish was likely related to retained sutures, which had sometimes served as a nidus for fungal growth. However, despite this finding, there was no demonstrated benefit from any of the treatments tested, including those cited for their anti-fungal and anti-bacterial properties. Notably, some of the treatments tested may be contraindicated for prophylactic use at the temperatures tested.
Although there were no statistical differences in survival observed among the treatment groups, fish exposed to hydrogen peroxide at all concentrations tested, consistently exhibited lower survival than the other treatments beginning at 24 h post-treatment. Toxic effects have been observed in other studies that exposed finfish to hydrogen peroxide at similar or higher concentrations for the purpose of preventing and treating fungal infection [29,31]. In these studies, as here, toxicity increased with increasing water temperature. Based on these observations, and despite its low regulatory priority by the U.S. Food and Drug Administration Table 7. a. Mixed Linear Effects modeling results (GLMM, using a logit link function) for 28 d survival. Randomeffects covariates included in all models were replicate tagging days and surgeons. Fixed-effect covariates tested against survival were: Treatment, fork length, weight, water temperature at tagging, presence of foreign material (0,1), sutures (0,1,2), suture tearing (0,1), inflammation or ulceration at suture site (0,1,2), and healing at suture site (1,2). See Table 1 for the full list of post-incision metrics. For 28-d survival we evaluated models at three stages: Variable at time of tagging, incision metrics after 7d for survivors, and 2 -way interactions among covariates. Only models with delta AIC values within approximately 2 units from the best model were included in the table, although the process included all subsets as possible models. b. Final GLMM linear mixed model.

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Evaluation of surgical incision healing and antiseptic use in Chinook salmon (Oncorhynchus tshawytscha) (FDA), extreme caution is warranted when subjecting finfish to hydrogen peroxide submersion at therapeutic levels. Furthermore, despite the widespread use of PolyAqua, and similar products that are promoted as health aids for bruised and lacerated fish, no evidence was found that it improved survival. It should be noted that very little is actually known about the effectiveness and safety of PolyAqua and similar products [78], and that although the difference in survival was not statistically significant between reference fish and treatment fish exposed to PolyAqua, the latter had lower survival.
Salt is also considered a low regulatory priority substance by the FDA [7] and is used in aquaculture as an inexpensive, readily available therapeutic. During our study of tag-effects on yearling and subyearling Chinook salmon [14,45], fish were transferred from freshwater to artificial seawater on holding day 15 (35 ppt; 11-13˚C). After transfer, these study fish experienced a decline in their rate of post-tagging mortality, indicating that the addition of salt may have been beneficial. However, during the current study, short-term post-operative salt baths did not significantly improve survival for treatment fish compared to the reference group.
Similarly, a one-time application of Argentyne to the surgical incisions of treatment fish did not improve survival or promote healing relative to reference fish.
Failure to impart a significant effect on wound healing or subsequent fungal growth may have been due to the relatively short duration of contact with Argentyne. Application times recommended for disinfecting eggs are significantly longer, at 10-15 minutes, and this treatment is typically repeated through time [48][49][50][51][52][53][54][55][56][57]. In a human surgical application, one study determined that a minimum contact time of 3 minutes was necessary for maximum disinfection of hands prior to conducting surgical procedures [79]. However, keeping fish out of water longer to prolong application time or actually immersing fish in Argentyne were both options expected to cause more harm than good. The presence of two secure ligatures at 7 d post-tagging was detrimental to fish survival through 28 d, based on comparisons of survival through time and on observations of the incision healing process. In some fish, the suture material also provided a nidus for fungal growth (also correlated with higher fish mortality) and ulcers were commonly observed beneath the suture material. Mechanistically, bacteria and/or fungi may have penetrated the dermal layer through these ulcers, leading to sepsis and subsequent death. No infectious agents were identified during the tag effects study [14] when inriver-migrant fish were recaptured after surgery and evaluated for gross and histological changes. However, of the subyearling Chinook salmon surgically implanted in 2007, only nine of 100 targeted were recaptured for necropsy. This dearth of subyearling recaptures was due to high mortality overall, so presumably, fish that were recaptured were the most robust of the tagged cohort.
Although this study found suture presence at seven days post-surgery was negatively correlated with survival, it is important to note that sutures can play an integral role in the healing process of surgical wounds in that they can neutralize tension at the incision site and promote tissue apposition. Booth (1985) described the ideal suture material as easy to handle and able to "maintain adequate tensile strength until its purpose is served". He further defined the ideal suture material as "nonelectrolytic, noncapillary, nonallergenic, and noncarcinogenic" [80].
For this study, properly tied sutures were expected to remain intact for approximately 2 weeks, the length of time anticipated for surgical incisions to functionally heal. Indeed, a respective 33.0 and 20.7% of incisions were not yet healed at 7 and 14 days post-surgery. This observation supports the need for suture presence beyond 7d to ensure optimal healing. Based on these findings, mitigation of suture impacts remains an important challenge.
In some cases, one can forego the use of sutures altogether; however, significant rates of tag loss may occur in study fish with no incision closure. Depending on the length, incisions without closure might also gape, thus exposing the body cavity to environmental pathogens or simply delaying healing due to poor tissue apposition. Braided or natural suture material may be chosen for its ability to break down faster than synthetic monofilament material, but these materials can potentially "wick" water and foreign material into the muscle layer [81][82][83][84][85][86][87]. Incision closure with alternative materials such as surgical glue carries a different suite of risks. Such closures can result in the incision reopening prematurely, can serve as a physical barrier to healing, and can lead to the formation of granulomas or fistulae [88][89][90][91].
Another option, which at first might seem counterintuitive, would be to use non-absorbable nylon to close surgical tag incisions. Non-absorbable nylon is characterized as having poor knot security relative to other suture material. As such, it tends to unravel and pull out. The authors have regularly observed this outcome, however, further study is needed before non-absorbable nylon can be considered as a preferred suture material. Such studies would include testing to determine the number of throws or extra knots needed in addition to the first knot placed in order to retain the ligature for an optimal period. This period would be long enough to promote healing but not long enough to serve as a nidus for foreign material. Future testing might also explore the use of one or more granny knots, which are prone to slippage, in lieu of standard square knots to secure each ligature.
The correlation between suture presence and the presence of fungi or fish mortality is increased when water temperatures exceeded 13.5˚C. Not surprisingly, overall survival and healing throughout the study was strongly and negatively correlated with increasing water temperature. For replicates 9 and 10, ambient river temperature (and thus handling and tagging temperature) exceeded 17˚C, and early post-tagging mortality was so high among all treatment replicates that none of them could be used in comparisons of survival at 28 d.
Other researchers have described warmer temperatures associated with delayed healing and inflammation at the incision site for surgically tagged fish [92,93]. These observations, along with those described here, serve as a reminder that surgical tagging of temperature-sensitive species at relatively high water temperatures may create significant and potentially lethal delayed effects. For Columbia River Chinook salmon, the reported temperature threshold for injecting PIT tags is 15˚C [94,95]. The results presented here indicated that an even lower maximum temperature threshold should be observed for surgical implantation of these fish.
For this study, Lower Granite Dam was used as the surgery site because of its suitability as a collection location for river-run fish, as well as its proximity to the Columbia River hydro-system, which was the primary study area. Unfortunately, the high temperatures at this location proved detrimental to survival during the river migration period of subyearling Chinook. To avoid seasonal temperature impacts in the future, implantation surgeries could either be conducted earlier in the year or in a laboratory or hatchery setting under more controlled environmental conditions. Surgically tagged fish could be held during most or all of the incision healing period, and their sutures could be removed prior to release. Of course, this practice could also confound the assumption that study fish were representative of their untagged cohorts.

Conclusion
Electronic transmitters have allowed researchers to study survival and movement of a variety of fish in situ since the mid-1950s [96]. However, for the validity of information collected through these types of studies, it is necessary to know that the behavior and survival of tagged fish is similar to that of their untagged cohorts. One way to approach this is through a paired release study such as described in tag effects study [14]. Laboratory studies can provide useful information on critical factors such as rates of tag loss, as well as providing insight into the healing process. However, readers are cautioned that results from laboratory studies can significantly underestimate the effects of surgical tagging compared to what may be actually observed in the field [14].
Comprehensive studies such as those described and referenced here will not be feasible for most situations where telemetry is used. Therefore, it is recommended that researchers and managers adopt a decidedly cautious approach to planning and interpretation of study outcomes that rely on telemetry tagging. Researchers will be well served to lean on the information collected and insights gained from previous studies [11,12,97], particularly those that have evaluated similar species, life stages, and environmental conditions.
The results of the study presented here specifically call into question the validity of data derived from surgical tagging of temperate species at temperatures greater than 13˚C. They also highlight the complications that can arise from the surgical process itself, such as retained sutures that may provide a nidus for fungi (or other foreign material). Unfortunately, an effective treatment that could be implemented during surgical tagging to prevent or mitigate these effects was not found. For fish migrating in cooler temperatures or reaching the ocean sooner, these surgical complications might never have occurred.
However, given similar conditions, the only feasible options that remain are tagging fish earlier in the migration season, tagging at locations where environmental conditions can be better controlled, foregoing the use of sutures altogether (i.e., opt for an injectable tag or a very small incision), or use suture material that has less knot security such as nylon. Photos from a surgeon with low survival relative to cohorts (a) and a surgeon with high survival relative to cohorts (b). Both photos were taken at the 7-d exam. Note the knots tied by the surgeon with low survival have remained intact over the first 7 d, as would be expected with proper knot tying technique. In comparison, knots tied by the surgeon with higher relative survival have loosened, with sutures pulling free from the incision. Photo b also illustrates suture tearing and the resulting wound perpendicular to the incision. (TIF) S1